JPH02417A - Transformation system - Google Patents

Abstract

PURPOSE: To enable effective transformation and convenient quick identification of oligomycin-resistance transformant strain of Penicillium chrysogenum, by carrying out transformation of Penicillium chrysogenum by using an oligomycin- resistance homologous gene as a selectable marker.

CONSTITUTION: A homologous DNA sequence encoding oligomycin resistance is incorporated into a genome of a transformant strain to give a transformant strain of Penicillium chrysogenum selectable in an oligomycin-containing medium. A gene (e.g. isopenicillin N synthase gene of Penicillium chrysogenum) of external part is expressed in Penicillium chrysogenum by the following method. Namely, (a) a host of Penicillium chrysogenum is formulated under transformation conditions with a recombinant vector comprising (i) the gene of external part and (ii) a homologous DNA sequence encoding oligomycin resistance, (b) an oligomycin-resistant colony is selected and (c) the gene is expressed in the host.

COPYRIGHT: (C)1990,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Industrial application field
illum chrysogenum)
The present invention relates to a transformation system for introducing P. chrysogenum, a method for producing and selecting transformed Benithulium chrysogenum using this transformation system, and a transformed strain of P. chrysogenum produced by the production method.

(b) Prior Art Penicillium chrysogenum is an industrially very important filamentous fungus because it produces penicillin G and penicillin V, which are important antibiotics. However, the development of recombinant DNA technology using P. chrysogenum as a host has hitherto been very difficult due to the lack of suitable loning vectors and transformation methods.

Identification of transformed cells, which are produced at low frequencies within P. chrysochenum, is difficult, if not impossible, without an appropriate selection system.

Transformation methods have been published for a number of filamentous fungi and generally depend on the availability of auxotrophic mutant strains. Then, the wild-type gene corresponding to this auxotrophic mutant strain is used to create a plasmid-produced form (plasmid-bor
ne rorm) provides a selectable name marker. A method for transforming P. chrysogenum using the above method was recently reported [Sanchez et al.
al), Gene, L987, 51@, 97-
On page 102, this method uses wild-type t to relieve a tryptophan-requiring mutant strain.
This method uses the rp-c gene. However, a problem with this method is that transformed strains can only be selected from strains that carry out auxotrophic mutations.

Reports of methods for transforming filamentous fungi using selectable genes for susceptibility have recently emerged. Aspergillus nidulans (
amd of Aspergillus-nidulans)
The s gene is derived from A. nidulans (A, n1dula).
ns) as well as other fungal species including P. chrysogenum [Beri and Turner, C.
currentGenetics, 1987, 11@
, pp. 639-641], in principle any species of filamentous fungi closely related to ascomycetes that lack the ability to grow on acetamide can be selected.

European Patent Application No. 0215539 and European Patent Application No. 0260
No. 762 et al. disclose a method for producing and selecting a transformed bacterial strain of the genus Penicillium. The latter document (unpublished at the time of the invention) vaguely mentions the possibility of using a gene conferring resistance to oligomycin in selecting transformed hosts. However, all of the examples concern the complementation of an auxotrophic host with structural genes encoding enzymes within metabolic pathways.

Oligomycin is a hydrophobic antibiotic that inhibits mitochondrial ATP synthase. Enns and Criddle, Arch.

Biochea+, Biophys, 1977, [Vol. 82, pp. 587-600.

Neurospora cr
A nuclear oligomain-resistant mutant of Assa assa contains altered subunit 9 (altered 5ubun).
it9) was proposed to contain an ATP synthase having the amino acid sequence of [Sebald et al.
ld etal), “Mi.10chondria 1
977-Genetics and Biogenesis
s or Mi. 10chondria″, Bandlow Y, et al.
'JA collection, double.

See Y. de Gruyter, Berlin]. The gene for subunit 9 of its altered oligomycin-resistant form was cloned from a mutant strain of Aspergillus nidulans [Ward et al.
rd et al), Mat, Gen, Gene
T, 1986, vol. 202, pp. 265-270]
, direct selection for oligomycin resistance was achieved after transforming wild-type A. nidulans.

However, to date, no method for transforming P. chrysogenum using a homologous gene for oligomycin resistance as a selectable marker has been published.

(c) Means and actions for solving the problem When the term "homologous" is used in this specification to refer to a DNA sequence that is similar to a gene, it is used to mean a gene or DNA sequence found in P. chrysogenum. It will be done.

This invention provides a method for transforming Penicillium chrysogenum and selecting a transformed strain produced, namely: I) a P. chrysogenum host organism and a recombinant vector having a homologous DNA sequence encoding oligomynon resistance; A method is provided comprising the steps of: mixing under transformation conditions and ii) selecting one or more microorganisms that grow when cultured in a medium containing oligomycin.

It is believed that the DNA sequence encoding oligomycin resistance was able to be integrated and expressed within P. chrysogenum.

In another aspect, the present invention provides a strain that is selectable in an oligomycin-containing medium, characterized in that the genome of the transformed strain has integrated therein a homologous DNA sequence encoding oligomycin resistance. It is clear from the above that a transformed strain of P. chrysogenum is provided.

An advantage of this invention is that it allows efficient transformation and convenient and rapid identification of oligomain-resistant transformed strains of P. chrysogenum.

The host cell may be any suitable strain of P. chrysogenum. For example, P. chrysogenum NRRL
1951 (wild type), or a nicotinamide auxotrophic strain of this wild type strain, e.g.
CM+ 317734 deposited with Mycological Institute (Mycological Institute).

Suitable recombinant vectors for use in this invention can be obtained by cloning the homologous DNA sequence encoding oligomycin resistance into any suitable vector, such as a plasmid or phage DNA.

Preferred vectors are plasmids, especially E.
Co11 origin of replication (E, Co11 origin or
Particular plasmids of this type include pUc9 (Viera and Messing, Gene, 1982, 19:259) and a sequence encoding a selectable phenotype in E. coli.
-268 pages).

Further suitable vectors include nismids, such as pHC79 (Horn and Collins, Gene, 1999).
(1980, Vol. 11, pp. 291-29i11).

In a preferred embodiment, a homologous DNA sequence encoding oligomycin resistance:
A portion of the chromosomal (nuclear) DNA of a mutant strain of Chrysogenum. Suitable homologous DNA sequences include those containing genes encoding proteins that inhibit the mechanism by which oligomycin exerts its fungicidal effect on untransformed P. chrysogenum hosts.

A suitable gene is a gene encoding an altered subunit of P. chrysogenum ATP-synthase, especially an altered subunit-9 protein.

A preferred DNA sequence carrying a gene encoding an altered subunit-9 protein of ATP-synthase of P. chrysogenum is produced by mutation of a nicotinamide auxotroph of a wild-type strain (B. chrysogenum NRRL 1951). Obtained from an oligomycin-resistant mutant strain of B. chrysogenum.

A mutant oligomycin-resistant bacterial strain that is particularly suitable for the above purpose is the strain bearing the name P. chrysogenum CMI314652, which was established in 1987 by the Commonwealth Mycological Institute under the provisions of the Futapest Treaty. It was deposited on April 16, 2013.

B. chrysogenum CMI 314652 forms the pond embodiment of this invention.

The origin and characteristics of P. chrysogenum CMI 314652 are as follows.

Origin of B. chrysogenum CMI 314652 P. chrysogenum CMI 317734 has undergone a spontaneous mutation that has made it resistant to oligomycin, providing an organism that can grow actively in a medium containing 5μ9/l of oligomycin. The mutant strain could not be distinguished from the parent strain in drug-free medium.

A heterokaryon test was performed to test that mutations are located in the nuclear genome [Rowlands and Turner, M.
o1. Gen, Genet, 1973, 1
See Vol. 26, pp. 201-216 (Methods) 2.

Taxonomy of P. chrysogenum CMI 314652 Wild-type strain of P. chrysogenum (NRRL 1
951) is manufactured by Northern Regional Research Laboratories, Bioria, Illinois, USA.
search Labora. 10ries), but [Leber and Alexander (Ra
Perand Alexander), Journa
l or the ElishaMitche! l 5
scientific 5ociety, 1945,
61, pp. 74-113], which is the parent strain of the strain used throughout the research in this application.

To obtain a DNA sequence encoding oligomycin resistance from the chromosomal DNA of a mutant oligomycin-resistant strain of B. chrysogenum, the DNA sequence can be probed (detectable) to isolate the appropriate tDNA fragment.
It is first necessary to construct a "library" of chromosomal DNA fragments.

This library can be produced by the following method (-L) Partial cleavage of chromosome D - < A with a suitable restriction enzyme, e.g. 5au3A: (b)! A suitable length is between 5 and 40kb,
Preferred is a size fractionation that provides fragments of 10-20 kb; (c) ligation of the fragment to a vector; and (d)
This is the transformation of an appropriate host with the generated recombinant DNA.

Any suitable vector can be used for library construction.

As a preferred embodiment of this invention, lambda vector EM
st, 4: Frischauret et al.
al), J, Mo1. Bio1, 1983,
170, pp. 827-842] was used as a vector to construct a library of chromosomal DNA fragments. 11131465
Insert the fragment obtained by size fractionating the chromosomal DNA of 2 into the old Ban site.

For all the above DNA treatments, standard methods can be used [Maniatis;
set al), +\Iolecular cloni
ng, ALabora. 10ry Manual”,
1932, C5HL Press, New York].

Any convenient host can be used for library construction. Preferred hosts include E. coli, e.g.
coli DHI, and this host is Hanahan (Hanahan).
han) method (J, Mo!, Biol.

(1933, 166@, pp. 567-580).

Clones within the library containing DNA sequences encoding oligomycin resistance can be identified by standard hybridization methods (see Maniatis et al., supra) using appropriate probes.

As probes, oligodeoxynucleotides having at least 13 bases are suitable, the sequence of which is complementary or partially complementary to a part of the known sequence of the gene encoding oligomycin resistance.

The DNA sequence encoding oligomycin resistance is
- When carrying a gene encoding fnidulans-9 protein with altered synthase, probes include stingray,
oligomycin resistance gene (oliC3
Suitable are those that are complementary to some of the known sequences of 1) (Ward and Turner, Mo1. Gen, Gen.
et., 1186, vol. 205, pp. 331-338). Alternatively, the probe may contain the entire o1iC31 gene, which can optionally be incorporated into a suitable vector.

A preferred probe is plasmid pMY11, which contains A. nidulans presubunit 9 DNA (Ward et al., Mo1. Gen, Genet.).

(1986, Vol. 202, pp. 265-270).

It will be appreciated that the probe will be labeled so that clones in the library containing complementary DNA can be easily identified.

The probe is usually radiolabeled, for example 32p.

Radioactive labels can be used by standard methods, such as less than 5"- or 3"-end labeling methods, including nick translating techniques.

Clones in the library that are "positive" (termed "positive" J (i.e., whose coveys have been found to hybridize with the probe) can be isolated, but oligomycin Resistance-encoding DNA can be obtained from this by cutting out with one or more appropriate restriction enzymes.

Aspects of the invention will be further clarified with reference to the following drawings.

Figure 1 (a) shows the restriction enzyme map of DNA (T) [one piece of chromosomal DNA of B. chrysogenum CMl 314652 + lambda EMBL4 vector DNA (full length 45 kb). ) [12.8 kb Hind m fragment derived from DNA (1)] Restriction enzyme map: 1 (C) shows DNA (III) [12.8 kb Hind m fragment derived from DNA (1)]
(II) derived from 9.3 kb old nd II[-EcoR
Restriction enzyme map of DNA (ff) [DNA ([1) or DNA (I]
Restriction enzyme map of the plasmid pPOLl constructed from DNA ([[) and pUC9;
) Figure shows restriction enzyme map of plasmid pPOL6 constructed with DNA ([[): Figure 1 (g) shows DNA
Plasmid p[! constructed with A(IV) and pUC9. c.
Restriction enzyme map of 8: Figure 2 shows five ρPOLI transformants (lanes 1 to 5) and P. chrysogenum CMl cut with EcoRI and probed with pUC9.
317734 (lane No. 6) by Southern electrophoresis pattern of total DNA; Figure 3(a) shows isopenicillin N chromosomal old Ban site (vertical arrow) and coding region (horizontal arrow).
Restriction enzyme map of the chromosomal region of the synthetase gene (V): Figure 3(b) shows the restriction enzyme map of plasmid pCYX4 constructed by cloning the 3.2 kb Xbol fragment of (V) into the 5alr site of puc9. Map: Figure 3 (C) is a restriction enzyme map of the plasmid ppo++ constructed from the RcoRI fragment of the pCYX4 insert subcloned into pPOL6; Figure 4 is the Southern electrophoresis pattern of the various samples listed below. Figure (margins below) PP011 transformed cells + l148 〃〃ko1144〃〃〃〃〃:1143 // //: I cut with hI11 old 142"": Cut with Xhol12421〃〃: BamHI'
E 2421 〃 ：E'co
RI'I 2421XhoI cleaved with B. chryzogesm C old 317734D NA
BamHI // // ttE
cORI 〃 //
//The above is a pC containing the isopenicillin synthetase-encoding region (V) shown in Figure 3(a).
Probed with YX4 EcoRI fragment.

In Figures 1 and 3, not all restriction sites are shown.

From the above, as a particularly preferred embodiment of this invention,
B. chrysogenum CM+ 314652 chromosome DN
It can be seen that the lambda EMBL4 library of A is probed with pMflI. The valuable piece of DNA obtained by this method was named of iC3, and the first
(a) Develop the restriction site arrangement shown in the figure.

Therefore, this invention uses DNA (t
), or a restriction fragment of DNA ([) containing the complete gene encoding oligomycin resistance (
However, this DNA is of the type that provides the restriction site arrangement shown in Figure (a).

In the method of this invention, the full-length DNA shown in FIG. 1(a) is used as the DNA encoding oligomycin resistance.
It is considered that using (1) is often inconvenient.

Therefore, if one has the complete gene encoding oligomycin resistance, it is preferred to use restriction fragments of DNA(r).

Preferred restriction fragments include the 12.8 kb old nd ■ fragment (II) with the restriction site arrangement shown in Figure 1(b), the 9.3 kb fragment (II) shown in Figure 1(c).
) find III-EcoRI fragment, and the 3.8 kb old nd l-Bam fragment shown in Figure 1(d).
Old fragments are listed.

Furthermore, as another 13 aspects of this invention, this invention: Yo, D
A recombinant vector consisting of a restriction fragment of NA (r) or DNA (1) carrying the complete gene encoding oligomycin resistance is provided.

Specific examples of recombinant vectors of this invention are as follows.

(a) pPOLl: A 15.5 kb plasmid constructed from DNA fragment (II) cloned into the former ndl11 site of pUC9 as defined above [1st
(e) See figure]: (b) pPOL6: DNA fragment cloned into the EcoR1 site of pUC9 as defined above (
A 12 kb plasmid constructed with I [[
) see figure]; and (c) pPOL8: a DNA fragment cloned as defined above into the EcoR1 site of pUC9 (
6.5kb plasmid [1st (g
) See figure.

In accordance with the provisions of the Budapest Treaty, E. coli (CSI strain) transformed with ppot,a was stored at the National Collections of Industrial and Marine Bacteria, Aberdeen, Scotland, UK.
Industrial and MarineBac
Jeria) on July 22, 1987, under deposit number: NCIB 12510.

In addition to the homologous DNA sequence encoding oligomycin resistance, suitable recombinant vectors for use in the methods of this invention typically contain one or more oligomycin resistance sequences that are desired to be produced by the transformed B. chrysogenum host. 1 that encodes a protein
It has another cloned gene. Such vectors form another aspect of this invention.

In yet another aspect, the present invention provides the following steps: (a) A recombinant befter comprising a B. chrysogenum host, i) an external gene, and ii) a homologous DNA sequence encoding oligomynon resistance. (b) selecting oligomycin-resistant colonies; and (c) expressing said gene in a host. It provides:

(Left below) The term "external genes" above is used to denote additional genes (homologous or heterologous) that are introduced into P. chrysogenum by the transformation methods described herein.

Additional cloned genes are introduced into recombinant vectors suitable for use in the methods of this invention by standard cloning techniques. Such a gene is cloned into a suitable vector as described above before the DNA sequence encoding oligomycin resistance is incorporated. However, these one or more additional cloned genes are advantageously introduced into a recombinant vector carrying a DNA sequence encoding oligomycin resistance.

A particular example of an additional cloned gene that may be present within the recombinant vectors described above is the isopenicillin N synthetase gene of B. chrysogenum.

A specific recombinant vector incorporating the isopenicillin N synthase gene is designated pPOIl and has the restriction site arrangement shown in Figure 3(c').

This isopenicillin N synthase gene is derived from Cephalosporium acremonium.
um acremonium) [Samson et al.
mson et at), Nature, 1985,
318@, pp. 191-194 or P. chrysogenum [Carr et al., CC Carr et ai], Gene, 1
986, Vol. 48, pp. 257-266] using synthetic oligonucleotides derived from the published sequence of the isopenicillin N synthetase gene of P. chrysogenum DN.
can be isolated by probing a library of A.

As is clear from the above, the present invention provides that the genome of a transformed strain contains (a) a homologous DNA sequence consisting of a gene encoding oligomycin resistance;
b) A transformed strain of P. chrysogenum which is selectable in an oligomycin-containing medium is provided, characterized in that it has integrated an additional gene which can be expressed in P. chrysogenum.

Preferred as the additional gene is the P. chrysogenum isopenicillin N synthetase gene, suitably isolated and cloned into a vector, as described above.

Suitable transformation conditions for use in the methods of this invention include those known in the art. for example,
Transformation can suitably be carried out in a medium containing calcium chloride and polyethylene glycol.

The strain of P. chrysogenum to be transformed is generally transformed to oligomycin resistance at a frequency of 1 to 5 times per μ9 DNA (1 to 10 transformants per viable protoplast). .

To select transformed protoplasts, selection is performed to regenerate all living protoplasts.
Usually about 2 hours after plating
Delay until 0 hours later.

Selection then selects oligomycin, generally about 3 μ9/I
This is done by covering with a medium containing Q.

Cultures are typically continued for 5 to 8 days at about 30°C. Transformed strains are selected and purified by streaking onto plates containing ligomycin.

(D) Implementation ρ1 The present invention will be explained by the following examples, but they are not intended to limit the invention.

The families and methods used in the examples described below are as follows.

(a) Strains P. chrysogenum CMI 317734: oligomycin-sensitive nicotinamide auxotroph, B. chrysogenum CMI 314652: nuclear oligomycin-resistant mutant derived from P. chrysogenum CM+ 317734, Enerychia coli auxotroph: DHI recA, p
a! A1hsr, hsm+, endo 1B, re
l^1 is Hanahan d (J, Mol.

Bio1, 1983, vol. 166, pp. 557-580)
transformed according to. MC1022araD1
39, (ara 1eu)7697, (lacZ)M2
S, galV, galK, 5LrA [Casadaban and Cohen
), J, Mo1. Bio1,.

1980, Vol. 138, pp. 179-207 is PuO2
Used for Totora [Viera and Messing, Gene, 19
1982, vol. 19, pp. 259-268.

(b) Plasmids The plasmid pWMII was used for probing, which was derived from Aspergillus nidulans (Ward et al.
Mo1. Gen, Genet, 1986, Vol. 202, pp. 265-270).

(c) DNA isolation and treatment A method for total extraction of B. chrysogenum DNA from mycelia and large-scale plasmid production was described by Ballance et al.
hem, Biophys, Res, Comm, , 1
The method described in 1983, Vol. 112, p. 284289 was used. Standard methods were used to process the DNA, including the small-scale plasmid single M method, reisolation of fragments from agarose gels, ligation, Southern methods, and lambda library plaque hybridization [Maniatis et al. −
Molecular Cloning: A Lab
ora. 10ry Manual” (19g2 years).

P. chrysogenum CMI for gene bank construction
Total DNA derived from 314652 was partially digested with 5au3A and size fractionated to obtain 10-20 kb fragments. Put these fragments into lambda vector E
'11BL4 into the Baff1 old cloning site (Flibshauf et al., J., 31o1.8io1.,
(1983, 170@, pp. 827-842).

(d) Cultivation and treatment of Penicillium genus strains A standard culture medium for Penicillium genus strains was used, supplemented with appropriate ζ nutrients by the drawing method. Oligomycin [Zig7-Chemical (Madhi)]
Sigma Che#1ical Co.

Ltd.), St. Louis, USA] was added at 1000 ton in ethanol. Collection of conidia and Aspergillus medium [complete Aspergillus medium (ACM) and minimal Aspergillus medium (AMX)
t)] is Mol by Rowlands and Turner.

Gen, GeneL, 1972, vol. 154, 3
This is similar to the one described on pages 11-317.

(e) Transformation of a Penicillium strain A suspension of fresh Penicillium strain spores (2Ra) was placed in a cal flask (D 200zf!).
]AMM and shaken at 25°C for about 16 hours. The resulting germinated conidia and young hyphae were collected by centrifugation (tabletop centrifuge), 0.9M! Wash with 1acI and wash with 5H/z (l) of Novozm 234 (Novozm 2
34) [Novo Industries (Nova 1
15. Contains Bugspurtco, Denmark. resuspended in 0.9M NaCQ of vc and gently shaken for 1-1.5 hours at 30°C, during which the degree of protoplastization was determined by microscopic examination of osmotically lysed samples. . After forming enough protoplasts (40-90%), a nylon filter cloth [Gallenkamp]
p), GMX-500J] and sintered glass (porosity 1), washed twice with 0.9M NaCC and once with IM sorbitol + 50mM CaCRz, and washed with IM sorbitol + 50mM NaCt! ! 0,5-5x 10' inside
/MQ variable @ turbidity and divided into 50 μQ each.

l-5μ9 DNA (10mM) Lis-HCl + 1mME
DTA, contained up to 5uQ at pH 7.5)
and then 25% PEG6000+50mMC
12.5 μl of a freshly made, filter-sterilized solution containing aCL ÷ l 0 mM Tris-HCl, pH 7.5 was added and mixed gently. After incubating on water for 20 minutes, 0.5 i (l) of the revised PEG solution was added. After incubating for 5 minutes at room temperature,
IM sorbitol + 50111MCaC (z) was added and mixed by inversion. Each aliquot was diluted with 0.9 MNa
The C77-containing molten ACM was mixed with lO sulfate and coated onto the ACM plate.

After 20 hours of incubation at 30°C, the plates were covered with ACh.E25xQ containing 3μ9/1 cyoligomycin. Cultivation was continued for an additional 5-8 hours at 30°C. The transformed colonies then begin to produce conidia on the surface of the agar.

Transformed cells were purified by streaking onto oligomycin-containing plates.

Aspergillus nidulans bra subunit 9
The plasmid pMYll containing the DNA was transformed into oligomycin-resistant P. chrysogenum 14 CM+31465.
Total DNA from Lambda EMBL4 was used as a probe for plaques of the lambda EMBL4 library. Stringency (5 trir + gency) that allows hybridization of sequences with approximately 73% homology (55°C, lX5S
When using C>, 6 plaques (about 20 in total) were found.
0006) hybridized strongly against a uniform weak background. Note that this frequency is consistent with the frequency expected if hybridization were assumed to be for a single copy of the nuclear gene.

For one of these clones, λoliC3,
A restriction enzyme map was created and analyzed. This map is the first (
a) Shown in the figure.

The 12kb old nd that grows the lambda DNA segment.
The fragment [Fig. 1(b)] was subcloned into pUC9 to form pPOLl [Fig. 1(e)]. Smaller plasmids pPOL6 and POL8
, the 9.3 kb old nd m -EcoRI fragment [Figure 1(f)] and 3.1! kb old nd iii
-Bam old fragment [Figure 1(g)] was created by subcloning each of the fragments.

(b) Transformation of Penicillium strain P. chrysogenum CM+ 317734 is 1 to 57
Transformed with oligomycin at a frequency of 10-5-10-μg DNA per viable protoplast.
All transformed strains tested showed increased resistance to oligomains when subcultured on selected media.

Selection of transformed protoplasts was delayed until about 20 hours after blating to regenerate all viable protoplasts before overlaying with oligomycin-containing medium. Transformed strains began to appear after 5-8 days.

Proliferation of a small percentage of the background of regenerated protoplasts occurs gradually, but P. chrysogenum C.
! On the plate where I11317734 was transformed with pUC9, some colonies that produced conidia were observed after 8 to 10 days. These colonies did not grow even after subculturing in oligomycin-containing medium, or pPO
When Ll was used to perform the transformation, similar colonies were observed to appear after 8-10 days.

(b) Phenotype of the transformed strain If a single copy of the resistance gene is inserted into the genome, one would expect to produce a semi-resistant transformed strain since the wild-type susceptible allele would still be present. Replacing the wild-type gene or inserting multiple copies of the resistance gene will result in a completely resistant phenotype. 7 tested
A total of three pPOLl-transformed bacterial cells were subcultured on oligomycin-containing plates and showed growth rates similar to P. chrysogenum CMI 314652.8. The remaining four showed semi-resistant growth rates. The fully resistant sector (π-shaped body) is similar to the sector that was stopped from being generated from a semi-resistant transformed strain of Aspergillus, and it is thought that genetic conversion has occurred (Ward et al., 5lo1. Gen, GeneL,.

(1986, Vol. 202, pp. 265-270), no completely resistant sector was observed. Plasmid pPO
L6 gave a fully resistant transformed strain of 577.
':.

Demonstrating integration of pPOLl Second, several transformed cell lines were analyzed for the presence of extraneous sequences by probing with pUc9 (Figure 2). Each transformed strain tested contained P from the donor plasmid pPOLl.
It has a UC arrangement.

Plasmid pPOL6 is a derivative of ppot,t with only one EcoRI site [Figure 1 ('')]. This site is linked to B. chrysogenum isopenicillin N.
3.2kb Ec containing the synthetase (IPS) gene
oRI fragment (3,2kb EcoRI-Xho
1 fragment) was inserted to create pPOIl [Figure 3(c)]. This gene was generated using a synthetic oligonucleotide derived from the sequence of the Cephalosporium acremonium gene (Samson et al., Nature,
(1985, Vol. 318, pp. 191-194) by probing the gene bank of B. chrysogenum DNA. The authenticity of the cloned fragments was confirmed by Karl et al., G.
This was confirmed by comparing with the B. chrysogenum IPS gene sequence published in EN%191116, Vol. 48, pp. 257-266. Bam1 (4 pPO1 cleaved with l
DNA from one transformed bacterium was analyzed using a 3.2 kb EcoR1 fragment containing the IPS-encoding region (Fig. 4) as a probe in the Southern method (Fig. 4). Among these transformed cells, +144
(fully resistant, track 2) and wild type (track 9)
It gives only the bands present within the DNA, probably generated by homologous replacement of the susceptible allele with the donor DNA. Transformed cells 1142°1143 and +148 (all semi-resistant) were transformed by another copy of the rPS sequence introduced by pPOIB) integration.
Gives specific hybridization bands (tracks 1.3 and 4). The pPO expected to form 11 with this probe
The BamH1 fragment of I1 is 1.3 kb and 6.0 kb.
It is a fragment of kb [Figure 3(c). This 7-ragment of Ba into the region encoding fPs
a present in all three transformants without integration of the old site, whereas the latter is 4.5k
It is reduced to band b and is also present in all three transformed cells. These two bands are stronger than the wild-type band and indicate multicopy integration (mult
icopy integration) may have occurred. Furthermore, 1142 and 1143 have weak hybridization bands of unpredictable size. DNA from another transformed cell +2421 was transformed into Xho1,
When cut with Bam old and EcoRl (semi-resistant, 5.
6 and 7 tracks), in addition to the wild type band (see tracks 8, 9 and 10) and the pond band, pP
Bands expected from the map of Oll [Xholnia, Oll
kb, EcoRl: 3.2 kb, Figure 3(c) is obtained.

These patterns match integrated I) Po11 sequences and are often multiplied and arranged by rearranged single Cobii sequences.

[Brief explanation of the drawing]

Figures 1 (a), 1 (b), 1 (c) and 1
(d) The diagram shows restriction enzyme maps of various restriction fragments,
(e) Diagram, I (1) Diagram and I (g) Diagram, restriction enzyme map of various plasmids, Figure 2, total D of various cells.
Figure 3 (a) is a restriction enzyme map of the chromosomal region of the IPS gene; Figure 3 (b) is a diagram of the migration pattern obtained by the NA Southern method;
) and Figure 3(c) are restriction enzyme maps of various plasmids, and Figure 4 is an electrophoresis pattern diagram of various transformed cells and DNA. □ −−P+ M −1−−Ippeng Coco
υ −mouth ω −−C force 2. ~---------
- ゝ-ゝ' ・Yoichi 567 Self 910 2.0 - 19- ・- Fig, A Procedural amendment (method) % formula % Ming Name Zed Transformation/Stem hli+1-, 4's, Middlesex, Brentford, Great West Road, Beecham House (no address) Representatives of Heacham Group LLC Representatives Decht Roberts 1 Person 1r:, Address: 530 3rd Quarter One Hill 5-1 Nishitenma, Kita-ku, Osaka City, t+Ii city order [1 attached December 20, 1983 [1 (Shipping IE) 2, o- 1, 0- 1' Fig, 4

Claims (1)

[Scope of Claims] 1. Penicillium chrysogenum that is selectable in an oligomycin-containing medium, characterized in that the genome of the transformed strain has incorporated therein a homologous DNA sequence encoding oligomycin resistance. Transformed strains. 2. Homologous DNA sequence is chromosome D of a mutant strain of Penicillium chrysogenum that has acquired oligomycin resistance
The transformed strain according to claim 1, which is a part of NA. 3. The transformed strain according to claim 1 or 2, wherein the DNA sequence encodes an altered subunit of ATP-silitase. 4. The transformed strain according to claim 3, wherein the altered subunit of ATP-silitase is subunit-9 protein. 5. The DNA sequence is Penicillium chrysogenum N.
The transformed strain according to claim 4, which is a DNA sequence derived from an oligomycin-resistant strain of Penicillium chrysogenum generated by mutation of the nicotinamide auxotrophic strain of RRL1951. 6. The DNA sequence is Penicillium chrysogenum C.
The transformed cell according to claim 5, which has a DNA sequence derived from MI314652. 7. The transformed bacterial strain according to any one of claims 1 to 6, wherein the genome of the transformed bacterial strain further contains an external gene. 8. The transformed strain according to claim 7, wherein the gene is the isopenicillin N synthetase gene of Penicillium chrysogenum. 9, a DNA segment designated oliC3, or o containing the complete gene encoding oligomycin resistance.
Recombinant vector carrying a restriction fragment of liC3. 10, pPOL1, pPOL6 and pPOL8. 11. The vector according to claim 9 or 10, comprising an additional cloned gene. 12. The vector according to claim 11, wherein the additional cloned gene is the Penicillium chrysogenum isopenicillin N synthetase gene. 13. A Penicillium chrysochenum strain transformed with the vector according to any one of claims 9 to 12. 14. Proliferation occurs when i) a Penicillium chrysochenum host organism and a recombinant vector carrying a homologous DNA sequence encoding oligomycin resistance are mixed under transformation conditions and ii) cultured in a medium containing oligomycin. A method for selecting a transformed strain produced by transforming Penicillium chrysogenum, which comprises the step of selecting one or more organisms that 15. Penicillium chrysogenum CMI 3146
52.